Important biological functions including maintenance of homeostasis in vivo, reproduction, development of individuals, metabolism, growth, control of the nervous, circulatory, immune, digestive or metabolic system, sensory adaptation, etc. are regulated by cells that receive endogenous factors such as various hormones and neurotransmitters or sensory stimulation like light or odor, via specific receptors present on cell membranes reserved for these factors or stimulation and interact with them. Many of these receptors for hormones or neurotransmitters by such functional regulation are coupled to guanine nucleotide-binding proteins (hereinafter, sometimes merely referred to as G proteins), and are characterized by developing a variety of functions through mediation of intracellular signal transduction via activation of the G proteins. In addition, these receptor proteins possess common seven transmembrane domains. Based on the foregoing, these receptors are thus collectively referred to as G protein-coupled receptors or seven transmembrane receptors. As such it is known that various hormones or neurotransmitters and their receptors are present and interact with each other to play important roles for regulating the biological functions. However, it often remains unclear if there are any other unknown substances (hormones, neurotransmitters, etc.) and receptors to these substances.
In the G protein-coupled receptors, ligands to some receptors that are subtypes having high homology in structure to known receptors may be readily predictable but in most cases, their endogenous ligands are unpredictable so that ligands corresponding to these receptors are hardly found. For this reason, these receptors are termed orphan receptors. It is likely that unidentified endogenous ligands to such orphan receptors would take part in biological phenomena poorly analyzed because the ligands were unknown. When such ligands are associated with important physiological effects or pathologic conditions, it is expected that development of these receptor agonists or antagonists will result in breakthrough new drugs (Stadel, J. et al., TiPS, 18, 430–437, 1997; Marchese, A. et al., TiPS, 20, 370–375, 1999; Civelli, O. et al., Brain Res., 848, 63–65, 1999, Howard, A. D. et al., TiPS, 22, 132–140, 2001).
Orphan receptors and ligands thereto often take part in a new physiological activity, and it is expected that their clarification will lead to development of new drugs. However, it is known that research on ligands to orphan receptors is accompanied by many difficulties. For example, it is generally unknown what secondary signal transduction system will take place after orphan receptors expressed on cells responded to ligands, and various response system should be examined. Moreover, tissues where ligands are present are not readily predictable so that various tissue extracts should be prepared. Furthermore, since an amount of ligand required to stimulate its receptor is sufficient even in an extremely low level when the ligand is a peptide, the amount of such a ligand present in vivo is a trace amount in many cases. In addition, a peptide is digested by peptidase to lose its activity, or undergoes non-specific adsorption so that its recovery becomes poor during purification. Thus, it is normally extremely difficult to extract such a ligand from the living body and isolate the necessary amount of the ligand for determination of its structure. The presence of many orphan receptors was unraveled, but only a very small part of the ligands to these receptors were discovered so far due to the foregoing problems.
GPR34 (SEQ ID NO: 1) (Genomics, 56, 12–21, 1999; Biochim. Biophys. Acta, 1446, 57–70, 1999), reported as an orphan G protein-coupled receptor, showed 26 to 31% homology to RSC338, RBintron, GPR23 or GPR17, which is a orphan receptor, and a poor (25% or less) homology to platelet-activating factor and uridine diphosphate glycoside, but is totally unknown for its ligand.
On the other hand, it is known that lysophosphatidylserine (hereinafter sometimes simply referred to as lyso-PS) has the histamine release activity on rat mast cells stimulated by an antigen or concanavalin A (Nature, 279, 250–252, 1979; FEBS Lett., 105, 58–62, 1979), the activity of releasing histamine by synergistically acting on rat mast cells together with nerve growth factor (NGF) (FEBS Lett., 138, 190–192, 1982), the growth regulating activity on human T cells (FEBS Lett., 316, 1–4, 1993), and the activity of potentiating the differentiation-inducing ability of NGF on PC12 cells (Neurosci. Lett., 248, 77–80, 1998). Since these actions are specific to lyso-PS and are observed in a relatively low level, it is predicted that specific receptors would mediate the actions. However, there is no report on identification of these receptors so far.
Phosphatidylserine (hereinafter sometimes simply referred to as PS) is known from old to inhibit blood coagulation or platelet agglutination response. Recently, it was demonstrated that PS was exposed on the cell surface of apoptotic cells and a receptor capable of recognizing PS is engaged in clearance of the apoptotic cells (Nature, 405, 85–90, 2000). However, the receptor is distinct from GPR34.
It has been desired to find a ligand to GPR34 and utilizing the screening system for pharmaceuticals using the ligand, thereby to develop pharmaceuticals having entirely a new mechanism of action.